rfc5089.IS-IS Protocol Extensions for Path Computation Element (PCE) Discovery

rfc5089.IS-IS Protocol Extensions for Path Computation Element (PCE) Discovery
rfc5089.IS-IS Protocol Extensions for Path Computation Element (PCE) Discovery

Network Working Group JL. Le Roux, Ed. Request for Comments: 5089 France Telecom Category: Standards Track JP. Vasseur, Ed. Cisco System Inc. Y. Ikejiri NTT Communications R. Zhang BT January 2008 IS-IS Protocol Extensions for

Path Computation Element (PCE) Discovery

Status of This Memo

This document specifies an Internet standards track protocol for the Internet community, and requests discussion and suggestions for

improvements. Please refer to the current edition of the "Internet

Official Protocol Standards" (STD 1) for the standardization state

and status of this protocol. Distribution of this memo is unlimited. Abstract

There are various circumstances where it is highly desirable for a

Path Computation Client (PCC) to be able to dynamically and

automatically discover a set of Path Computation Elements (PCEs),

along with information that can be used by the PCC for PCE selection. When the PCE is a Label Switching Router (LSR) participating in the

Interior Gateway Protocol (IGP), or even a server participating

passively in the IGP, a simple and efficient way to announce PCEs

consists of using IGP flooding. For that purpose, this document

defines extensions to the Intermediate System to Intermediate System (IS-IS) routing protocol for the advertisement of PCE Discovery

information within an IS-IS area or within the entire IS-IS routing

domain.

Le Roux, et al. Standards Track [Page 1]

Table of Contents

1. Introduction (2)

2. Terminology (4)

3. Overview (5)

3.1. PCE Discovery Information (5)

3.2. Flooding Scope (5)

4. The IS-IS PCED Sub-TLV (5)

4.1. PCE-ADDRESS Sub-TLV (6)

4.2. The PATH-SCOPE Sub-TLV (7)

4.3. PCE-DOMAIN Sub-TLV (9)

4.4. NEIG-PCE-DOMAIN Sub-TLV (10)

4.5. PCE-CAP-FLAGS Sub-TLV (10)

5. Elements of Procedure (11)

6. Backward Compatibility (12)

7. IANA Considerations (12)

8. Security Considerations (12)

9. Manageability Considerations (13)

9.1. Control of Policy and Functions (13)

9.2. Information and Data Model (13)

9.3. Liveness Detection and Monitoring (13)

9.4. Verify Correct Operations (13)

9.5. Requirements on Other Protocols and Functional

Components (13)

9.6. Impact on Network Operations (14)

10. Acknowledgments (14)

11. References (15)

11.1. Normative References (15)

11.2. Informative References (15)

1. Introduction

[RFC4655] describes the motivations and architecture for a Path

Computation Element (PCE)-based path computation model for

Multi-Protocol Label Switching (MPLS) and Generalized MPLS (GMPLS)

Traffic Engineered Label Switched Paths (TE LSPs). The model allows for the separation of the PCE from a Path Computation Client (PCC)

(also referred to as a non co-located PCE) and allows for cooperation between PCEs (where one PCE acts as a PCC to make requests of the

other PCE). This relies on a communication protocol between a PCC

and PCE, and also between PCEs. The requirements for such a

communication protocol can be found in [RFC4657], and the

communication protocol is defined in [PCEP].

The PCE architecture requires that a PCC be aware of the location of one or more PCEs in its domain, and, potentially, of PCEs in other

domains, e.g., in the case of inter-domain TE LSP computation.

Le Roux, et al. Standards Track [Page 2]

A network may contain a large number of PCEs, each with potentially

distinct capabilities. In such a context, it is highly desirable to have a mechanism for automatic and dynamic PCE discovery that allows PCCs to automatically discover a set of PCEs, along with additional

information about each PCE that may be used by a PCC to perform PCE

selection. Additionally, it is valuable for a PCC to dynamically

detect new PCEs, failed PCEs, or any modification to the PCE

information. Detailed requirements for such a PCE discovery

mechanism are provided in [RFC4674].

Note that the PCE selection algorithm applied by a PCC is out of the scope of this document.

When PCCs are LSRs participating in the IGP (OSPF or IS-IS), and PCEs are either LSRs or servers also participating in the IGP, an

effective mechanism for PCE discovery within an IGP routing domain

consists of utilizing IGP advertisements.

This document defines extensions to IS-IS [ISO] to allow a PCE in an IS-IS routing domain to advertise its location, along with some

information useful to a PCC for PCE selection, so as to satisfy

dynamic PCE discovery requirements set forth in [RFC4674].

Generic capability advertisement mechanisms for IS-IS are defined in [RFC4971]. These allow a router to advertise its capabilities within an IS-IS area or an entire IS-IS routing domain. This document

leverages this generic capability advertisement mechanism to fully

satisfy the dynamic PCE discovery requirements.

This document defines a new sub-TLV (named the PCE Discovery (PCED)) to be carried within the IS-IS Router Capability TLV ([RFC4971]).

The PCE information advertised is detailed in Section 3. Protocol

extensions and procedures are defined in Sections 4 and 5.

The IS-IS extensions defined in this document allow for PCE discovery within an IS-IS routing domain. Solutions for PCE discovery across

AS boundaries are beyond the scope of this document, and are for

further study.

This document defines a set of sub-TLVs that are nested within each

other. When the degree of nesting TLVs is 2 (a TLV is carried within another TLV) the TLV carried within a TLV is called a sub-TLV.

Strictly speaking, when the degree of nesting is 3, a sub-sub-TLV is carried within a sub-TLV that is itself carried within a TLV. For

the sake of terminology simplicity, a TLV carried within another TLV is called a sub-TLV regardless of the degree of nesting.

Le Roux, et al. Standards Track [Page 3]

2. Terminology

ABR: IS-IS Area Border Router.

AS: Autonomous System.

IGP: Interior Gateway Protocol. Either of the two routing protocols, Open Shortest Path First (OSPF) or Intermediate System to

Intermediate system (IS-IS).

Intra-area TE LSP: A TE LSP whose path does not cross an IGP area

boundary.

Intra-AS TE LSP: A TE LSP whose path does not cross an AS boundary.

Inter-area TE LSP: A TE LSP whose path transits two or more IGP

areas. That is, a TE LSP that crosses at least one IGP area

boundary.

Inter-AS TE LSP: A TE LSP whose path transits two or more ASes or

sub-ASes (BGP confederations). That is, a TE LSP that crosses at

least one AS boundary.

IS-IS LSP: Link State PDU.

LSR: Label Switching Router.

PCC: Path Computation Client. Any client application requesting a

path computation to be performed by a Path Computation Element.

PCE: Path Computation Element. An entity (component, application, or network node) that is capable of computing a network path or route

based on a network graph and applying computational constraints.

PCED: PCE Discovery.

PCE-Domain: In a PCE context, this refers to any collection of

network elements within a common sphere of address management or path computational responsibility (referred to as a "domain" in

[RFC4655]). Examples of PCE-Domains include IGP areas and ASes.

This should be distinguished from an IS-IS routing domain as defined by [ISO].

PCEP: Path Computation Element communication Protocol.

TE LSP: Traffic Engineered Label Switched Path.

TLV: Type-Length-Variable data encoding.

Le Roux, et al. Standards Track [Page 4]

The key words "MUST", "MUST NOT", "REQUIRED", "SHALL", "SHALL NOT",

"SHOULD", "SHOULD NOT", "RECOMMENDED", "MAY", and "OPTIONAL" in this document are to be interpreted as described in [RFC2119].

3. Overview

3.1. PCE Discovery Information

The PCE discovery information is composed of:

- The PCE location: an IPv4 and/or IPv6 address that is used to

reach the PCE. It is RECOMMENDED to use an address that is always reachable if there is any connectivity to the PCE;

- The PCE path computation scope (i.e., intra-layer, inter-area,

inter-AS, or inter-layer);

- The set of one or more PCE-Domain(s) into which the PCE has

visibility and for which the PCE can compute paths;

- The set of zero, one, or more neighbor PCE-Domain(s) toward which the PCE can compute paths;

- A set of communication capabilities (e.g., support for request

prioritization) and path computation-specific capabilities (e.g., supported constraints).

PCE discovery information is, by nature, fairly static and does not

change with PCE activity. Changes in PCE discovery information may

occur as a result of PCE configuration updates, PCE

deployment/activation, PCE deactivation/suppression, or PCE failure. Hence, this information is not expected to change frequently.

3.2. Flooding Scope

The flooding scope for PCE information advertised through IS-IS can

be a single L1 area, an L1 area and the L2 sub-domain, or the entire IS-IS routing domain.

4. The IS-IS PCED Sub-TLV

The IS-IS PCED sub-TLV contains a non-ordered set of sub-TLVs.

The format of the IS-IS PCED sub-TLV and its sub-TLVs is identical to the TLV format used by the Traffic Engineering Extensions to IS-IS

[RFC3784]. That is, the TLV is comprised of 1 octet for the type, 1 octet specifying the TLV length, and a value field. The Length field defines the length of the value portion in octets.

Le Roux, et al. Standards Track [Page 5]

TYPE: 5

LENGTH: Variable

VALUE: Set of sub-TLVs

Five sub-TLVs are defined:

Sub-TLV type Length Name

1 variable PCE-ADDRESS sub-TLV

2 3 PATH-SCOPE sub-TLV

3 variable PCE-DOMAIN sub-TLV

4 variable NEIG-PCE-DOMAIN sub-TLV

5 variable PCE-CAP-FLAGS sub-TLV

The PCE-ADDRESS and PATH-SCOPE sub-TLVs MUST always be present within the PCED sub-TLV.

The PCE-DOMAIN and NEIG-PCE-DOMAIN sub-TLVs are optional. They MAY

be present in the PCED sub-TLV to facilitate selection of

inter-domain PCEs.

The PCE-CAP-FLAGS sub-TLV is optional and MAY be present in the PCED sub-TLV to facilitate the PCE selection process.

Any unrecognized sub-TLV MUST be silently ignored.

The PCED sub-TLV is carried within an IS-IS CAPABILITY TLV defined in [RFC4971].

No additional sub-TLVs will be added to the PCED TLV in the future.

If a future application requires the advertisement of additional PCE information in IS-IS, this will not be carried in the CAPABILITY TLV.

The following sub-sections describe the sub-TLVs that may be carried within the PCED sub-TLV.

4.1. PCE-ADDRESS Sub-TLV

The PCE-ADDRESS sub-TLV specifies an IP address that can be used to

reach the PCE. It is RECOMMENDED to make use of an address that is

always reachable, provided the PCE is alive and reachable.

The PCE-ADDRESS sub-TLV is mandatory; it MUST be present within the

PCED sub-TLV. It MAY appear twice, when the PCE has both an IPv4 and IPv6 address. It MUST NOT appear more than once for the same address type. If it appears more than once for the same address type, only

the first occurrence is processed and any others MUST be ignored.

Le Roux, et al. Standards Track [Page 6]

TYPE: 1

LENGTH: 5 for an IPv4 address or 17 for an IPv6 address.

VALUE: This comprises one octet indicating the address-type and 4 or 16 octets encoding the IPv4 or IPv6 address to be used to reach the PCE.

Address-type:

1 IPv4

2 IPv6

4.2. The PATH-SCOPE Sub-TLV

The PATH-SCOPE sub-TLV indicates the PCE path computation scope,

which refers to the PCE’s ability to compute or take part in the

computation of paths for intra-area, inter-area, inter-AS, or

inter-layer TE LSPs.

The PATH-SCOPE sub-TLV is mandatory; it MUST be present within the

PCED sub-TLV. There MUST be exactly one instance of the PATH-SCOPE

sub-TLV within each PCED sub-TLV. If it appears more than once only the first occurrence is processed and any others MUST be ignored.

The PATH-SCOPE sub-TLV contains a set of bit flags indicating the

supported path scopes, and four fields indicating PCE preferences.

The PATH-SCOPE sub-TLV has the following format:

TYPE: 2

LENGTH: 3

VALUE: This comprises a 1-octet flags field where each flag

represents a supported path scope, followed by a 2-octet

preferences field indicating PCE preferences.

Here is the structure of the flags field:

+-+-+-+-+-+-+-+-+

|0|1|2|3|4|5|Res|

+-+-+-+-+-+-+-+-+

Le Roux, et al. Standards Track [Page 7]

Bit Path Scope

0 L bit: Can compute intra-area paths.

1 R bit: Can act as PCE for inter-area TE LSP computation.

2 Rd bit: Can act as a default PCE for inter-area TE LSP

computation.

3 S bit: Can act as PCE for inter-AS TE LSP computation.

4 Sd bit: Can act as a default PCE for inter-AS TE LSP

computation.

5 Y bit: Can act as PCE for inter-layer TE LSP

computation.

6-7 Reserved for future use.

Here is the structure of the preferences field:

+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+

|PrefL|PrefR|PrefS|PrefY| Res |

+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+

PrefL field: PCE’s preference for intra-area TE LSP computation.

PrefR field: PCE’s preference for inter-area TE LSP computation.

PrefS field: PCE’s preference for inter-AS TE LSP computation.

Pref-Y field: PCE’s preference for inter-layer TE LSP computation.

Res: Reserved for future use.

The L, R, S, and Y bits are set when the PCE can act as a PCE for

intra-area, inter-area, inter-AS, or inter-layer TE LSP computation, respectively. These bits are non-exclusive.

When set, the Rd bit indicates that the PCE can act as a default PCE for inter-area TE LSP computation (that is, the PCE can compute a

path toward any neighbor area). Similarly, when set, the Sd bit

indicates that the PCE can act as a default PCE for inter-AS TE LSP

computation (the PCE can compute a path toward any neighbor AS).

When the Rd and Sd bit are set, the PCED sub-TLV MUST NOT contain a

NEIG-PCE-DOMAIN sub-TLV (see Section 4.4).

When the R bit is clear, the Rd bit SHOULD be clear on transmission

and MUST be ignored on receipt. When the S bit is clear, the Sd bit SHOULD be clear on transmission and MUST be ignored on receipt.

The PrefL, PrefR, PrefS and PrefY fields are each three bits long and allow the PCE to specify a preference for each computation scope,

Le Roux, et al. Standards Track [Page 8]

where 7 reflects the highest preference. Such preferences can be

used for weighted load balancing of path computation requests. An

operator may decide to configure a preference for each computation

scope at each PCE so as to balance the path computation load among

them. The algorithms used by a PCC to balance its path computation

requests according to such PCE preferences are out of the scope of

this document and are a matter for local or network-wide policy. The same or different preferences may be used for each scope. For

instance, an operator that wants a PCE capable of both inter-area and inter-AS computation to be preferred for use for inter-AS

computations may configure PrefS higher than PrefR.

When the L, R, S, or Y bits are cleared, the PrefL, PrefR, PrefS, and PrefY fields SHOULD respectively be set to 0 on transmission and MUST be ignored on receipt.

Both reserved fields SHOULD be set to zero on transmission and MUST

be ignored on receipt.

4.3. PCE-DOMAIN Sub-TLV

The PCE-DOMAIN sub-TLV specifies a PCE-Domain (area and/or AS) where the PCE has topology visibility and through which the PCE can compute paths.

The PCE-DOMAIN sub-TLV SHOULD be present when PCE-Domains for which

the PCE can operate cannot be inferred by other IGP information: for instance, when the PCE is inter-domain capable (i.e., when the R bit or S bit is set) and the flooding scope is the entire routing domain (see Section 5 for a discussion of how the flooding scope is set and interpreted).

A PCED sub-TLV may include multiple PCE-DOMAIN sub-TLVs when the PCE has visibility into multiple PCE-Domains.

The PCE-DOMAIN sub-TLV has the following format:

TYPE: 3

LENGTH: Variable

VALUE: This is composed of one octet indicating the domain-type

(area ID or AS Number) and a variable length IS-IS area ID or a 32-bit AS number, identifying a PCE-Domain where the PCE has visibility and can compute paths.

Two domain types are defined:

1 Area ID

2 AS Number

Le Roux, et al. Standards Track [Page 9]

The Area ID is the area address as defined in [ISO].

When the AS number is coded in two octets, the AS Number field MUST

have its first two octets set to 0.

4.4. NEIG-PCE-DOMAIN Sub-TLV

The NEIG-PCE-DOMAIN sub-TLV specifies a neighbor PCE-Domain (area or AS) toward which a PCE can compute paths. It means that the PCE can take part in the computation of inter-domain TE LSPs with paths that transit this neighbor PCE-Domain.

A PCED sub-TLV may include several NEIG-PCE-DOMAIN sub-TLVs when the PCE can compute paths towards several neighbor PCE-Domains.

The NEIG-PCE-DOMAIN sub-TLV has the same format as the PCE-DOMAIN

sub-TLV:

TYPE: 4

LENGTH: Variable

VALUE: This comprises one octet indicating the domain-type (area ID or AS Number) and a variable length IS-IS area ID or a 32-bit AS number, identifying a PCE-Domain toward which

the PCE can compute paths.

Two domain types are defined:

1 Area ID

2 AS Number

The Area ID is the area address as defined in [ISO].

When the AS number is coded in two octets, the AS Number field MUST

have its first two octets set to 0.

The NEIG-PCE-DOMAIN sub-TLV MUST be present at least once with

domain-type set to 1 if the R bit is set and the Rd bit is cleared,

and MUST be present at least once with domain-type set to 2 if the S bit is set and the Sd bit is cleared.

4.5. PCE-CAP-FLAGS Sub-TLV

The PCE-CAP-FLAGS sub-TLV is an optional sub-TLV used to indicate PCE capabilities. It MAY be present within the PCED sub-TLV. It MUST

NOT be present more than once. If it appears more than once, only

the first occurrence is processed and any others MUST be ignored.

Le Roux, et al. Standards Track [Page 10]

The value field of the PCE-CAP-FLAGS sub-TLV is made up of an array

of units of 32-bit flags numbered from the most significant bit as

bit zero, where each bit represents one PCE capability.

The PCE-CAP-FLAGS sub-TLV has the following format:

TYPE: 5

LENGTH: Multiple of 4

VALUE: This contains an array of units of 32-bit flags numbered

from the most significant as bit zero, where each bit

represents one PCE capability.

The PCE capability registry is managed by IANA; it is common with

OSPF and defined in [RFC5088].

Reserved bits SHOULD be set to zero on transmission and MUST be

ignored on receipt.

5. Elements of Procedure

The PCED sub-TLV is advertised within an IS-IS Router Capability TLV defined in [RFC4971]. As such, elements of procedures are inherited from those defined in [RFC4971].

The flooding scope is controlled by the S flag in the IS-IS Router

Capability TLV (see [RFC4971]). When the scope of the PCED sub-TLV

is area local, it MUST be carried within an IS-IS Router Capability

TLV having the S bit cleared. When the scope of the PCED sub-TLV is the entire IS-IS routing domain, it MUST be carried within an IS-IS

Router Capability TLV having the S bit set. Note that when only the L bit of the PATH-SCOPE sub-TLV is set, the flooding scope MUST be

area local.

Note that an L1L2 node may include a PCED TLV in a Router Capability TLV with the S bit cleared in both in its L1 and L2 LSPs. This

allows the flooding scope to be restricted to the L1 area and the L2 sub-domain.

When the PCE function is deactivated, the IS-IS speaker advertising

this PCE MUST originate a new IS-IS LSP that no longer includes the

corresponding PCED TLV.

The PCE address (i.e., the address indicated within the PCE-ADDRESS

sub-TLV) SHOULD be reachable via some prefixes advertised by IS-IS.

The PCED sub-TLV information regarding a specific PCE is only

considered current and useable when the router advertising this

Le Roux, et al. Standards Track [Page 11]

information is itself reachable via IS-IS calculated paths at the

level of the LSP in which the PCED sub-TLV appears.

A change in the state of a PCE (activate, deactivate, parameter

change) MUST result in a corresponding change in the PCED sub-TLV

information advertised by an IS-IS router (inserted, removed,

updated) in its LSP. The way PCEs determine the information they

advertise, and how that information is made available to IS-IS, is

out of the scope of this document. Some information may be

configured (e.g., address, preferences, scope) and other information may be automatically determined by the PCE (e.g., areas of

visibility).

A change in information in the PCED sub-TLV MUST NOT trigger any SPF computation at a receiving router.

6. Backward Compatibility

The PCED sub-TLV defined in this document does not introduce any

interoperability issues.

An IS-IS router not supporting the PCED sub-TLV will just silently

ignore the sub-TLV as specified in [RFC4971].

7. IANA Considerations

IANA has defined a registry for the sub-TLVs carried in the IS-IS

Router Capability TLV defined in [RFC4971]. IANA has assigned a new sub-TLV codepoint for the PCED sub-TLV carried within the Router

Capability TLV.

Value Sub-TLV References

----- -------- ----------

5 PCED sub-TLV (this document)

8. Security Considerations

This document defines IS-IS extensions for PCE discovery within an

administrative domain. Hence the security of the PCE discovery

relies on the security of IS-IS.

Mechanisms defined to ensure authenticity and integrity of IS-IS LSPs [RFC3567] and their TLVs, can be used to secure the PCED sub-TLV as

well.

IS-IS provides no encryption mechanism for protecting the privacy of LSPs and, in particular, the privacy of the PCE discovery

information.

Le Roux, et al. Standards Track [Page 12]

9. Manageability Considerations

Manageability considerations for PCE Discovery are addressed in

Section 4.10 of [RFC4674].

9.1. Control of Policy and Functions

Requirements for the configuration of PCE discovery parameters on

PCCs and PCEs are discussed in Section 4.10.1 of [RFC4674].

In particular, a PCE implementation SHOULD allow the following

parameters to be configured on the PCE:

-The PCE IPv4/IPv6 address(es) (see Section 4.1).

-The PCE Scope, including the inter-domain functions (inter-area, inter-AS, inter-layer), the preferences, and whether the PCE can act as default PCE (see Section 4.2).

-The PCE-Domains (see Section 4.3).

-The neighbor PCE-Domains (see Section 4.4).

-The PCE capabilities (see Section 4.5).

9.2. Information and Data Model

A MI

B module for PCE Discovery is defined in [PCED-MIB].

9.3. Liveness Detection and Monitoring

This document specifies the use of IS-IS as a PCE Discovery Protocol. The requirements specified in [RFC4674] include the ability to

determine liveness of the PCE Discovery protocol. Normal operation

of the IS-IS protocol meets these requirements.

9.4. Verify Correct Operations

The correlation of information advertised against information

received can be achieved by comparing the information in the PCED

sub-TLV received by the PCC with that stored at the PCE using the

PCED MIB [PCED-MIB]. The number of dropped, corrupt, and rejected

information elements are available through the PCED MIB.

9.5. Requirements on Other Protocols and Functional Components

The IS-IS extensions defined in this document do not imply any

requirements on other protocols.

Le Roux, et al. Standards Track [Page 13]

9.6. Impact on Network Operations

Frequent changes in PCE information advertised in the PCED sub-TLV

may have a significant impact on IS-IS and might destabilize the

operation of the network by causing the PCCs to swap between PCEs.

As discussed in Section 4.10.4 of [RFC4674], it MUST be possible to

apply at least the following controls:

- Configurable limit on the rate of announcement of changed

parameters at a PCE.

- Control of the impact on PCCs, such as through rate-limiting

the processing of PCED sub-TLVs.

- Configurable control of triggers that cause a PCC to swap to

another PCE.

10. Acknowledgments

We would like to thank Lucy Wong, Adrian Farrel, Les Ginsberg, Mike

Shand, Lou Berger, David Ward, Ross Callon, and Lisa Dusseault for

their useful comments and suggestions.

Le Roux, et al. Standards Track [Page 14]

11. References

11.1. Normative References

[ISO] "Intermediate System to Intermediate System Intra-Domain Routeing Exchange Protocol for use in Conjunction with

the Protocol for Providing the Connectionless-mode

Network Service" ISO/IEC 10589:2002 Second Edition.

[RFC2119] Bradner, S., "Key words for use in RFCs to Indicate

Requirement Levels", BCP 14, RFC 2119, March 1997.

[RFC3567] Li, T. and R. Atkinson, "Intermediate System to

Intermediate System (IS-IS) Cryptographic

Authentication", RFC 3567, July 2003.

[RFC3784] Smit, H. and T. Li, "Intermediate System to Intermediate System (IS-IS) Extensions for Traffic Engineering (TE)", RFC 3784, June 2004.

[RFC4971] Vasseur, JP., Ed., Shen, N., Ed., and R. Aggarwal, Ed., "Intermediate System to Intermediate System (IS-IS)

Extensions for Advertising Router Information", RFC

4971, July 2007.

[RFC5088] Le Roux, JL., Ed., Vasseur, JP., Ed., Ikejiri, Y., and

R. Zhang, "OSPF Protocol Extensions for Path Computation Element (PCE) Discovery", RFC 5088, January 2008.

11.2. Informative References

[PCED-MIB] Stephan, E., "Definitions of Managed Objects for Path

Computation Element Discovery", Work in Progress, March 2007.

[PCEP] Vasseur, JP., Ed., and JL. Le Roux, Ed., "Path

Computation Element (PCE) communication Protocol (PCEP) ", Work in Progress, November 2007.

[RFC4655] Farrel, A., Vasseur, JP., and J. Ash, "A Path

Computation Element (PCE)-Based Architecture", RFC 4655, August 2006.

[RFC4657] Ash, J., Ed., and J. Le Roux, Ed., "Path Computation

Element (PCE) Communication Protocol Generic

Requirements", RFC 4657, September 2006.

Le Roux, et al. Standards Track [Page 15]

[RFC4674] Le Roux, J., Ed., "Requirements for Path Computation

Element (PCE) Discovery", RFC 4674, October 2006. Authors’ Addresses

Jean-Louis Le Roux (Editor)

France Telecom

2, avenue Pierre-Marzin

22307 Lannion Cedex

FRANCE

EMail: jeanlouis.leroux@https://www.360docs.net/doc/447393216.html,

Jean-Philippe Vasseur (Editor)

Cisco Systems, Inc.

1414 Massachusetts avenue

Boxborough, MA 01719

USA

EMail: jpv@https://www.360docs.net/doc/447393216.html,

Yuichi Ikejiri

NTT Communications Corporation

1-1-6, Uchisaiwai-cho, Chiyoda-ku

Tokyo 100-8019

JAPAN

EMail: y.ikejiri@https://www.360docs.net/doc/447393216.html,

Raymond Zhang

BT

2160 E. Grand Ave.

El Segundo, CA 90025

USA

EMail: raymond.zhang@https://www.360docs.net/doc/447393216.html,

Le Roux, et al. Standards Track [Page 16]

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Le Roux, et al. Standards Track [Page 17]

蛋白的纯化

第二部分:蛋白的纯化 如何区分蛋白表达在上清还是包涵体? 破碎细胞后离心分别收集上清和沉淀,表达的蛋白可能分布在上清中也有可能分布在沉淀中,还有可能是二者中都有分布。 根据我们实验室的经验,超声碎菌之后,如果菌液比较清亮,沉淀比较少,那表达的蛋白基本上是可溶的。但如果超声完之后,菌液是浑浊的,而且当离心之后,离下的沉淀比较多,而且沉淀的颜色也比较白,那基本上就是包涵体了。包涵体是基因重组蛋白在大肠杆菌中高水平表达时所形成的无活性的蛋白质聚集体,难溶于氺,可溶于变性剂如尿素,盐酸胍等,其实,包涵体也就是我们常说的不可溶蛋白。对于后者,可将上清和沉淀分别跑一个PAGE,看看上清中的量能达到多少,对于某些蛋白来说,一部分是以包涵体形式表达,一部分是以可溶的形式表达,而且量也不少,可以满足后续实验的需要,这个时候最好是纯可溶的,因为包涵体即使最后复性,活性也不太可信。 对于沉淀跑SDS-PAGE,如何处理,用什么使其溶解,还有在大肠杆菌中表达的蛋白,在提取过程中,使用什么蛋白提取缓冲液。 沉淀用Buffer B重悬,(组成:8M尿素+10mMTRIS base+100mM NaH2PO4,用NaOH调节pH到8.0),1克沉淀(湿重)加5ml Buffer B,使其充分溶解(可以放在微量震荡器上震荡20min),然后室温下12000转离心20min,留上清,弃沉淀。 取10ul上清加入10ul 2xSDS上样缓冲液,就可以跑PAGE了。 无论是纯可溶蛋白还是包涵体,在菌体裂解这一步我用的都是Lysis Buffer(组成:10mM 咪唑+300mM NaCl+50mM NaH2PO4,用NaOH调节pH到8.0)每克菌体(湿重)加2-5ml Lysis Buffer,充分悬起后,加入溶菌酶4度作用半小时就可以超声破碎了。 包涵体,简单的说就是翻译的蛋白没有正确折叠而聚集在一起形成的,主要的是疏水作用。实际上就是很多个蛋白分子,这些蛋白并不是交联在一起的,用高浓度的尿素和盐酸胍可以使他们变性,解聚。 电泳检测的话,可以用SDS-PAGE检测,在上样之前,需要用上样缓冲液处理样品,处理后,包涵体也就解聚了,每个蛋白分子与SDS结合,形成了可溶物。 包涵体是不容易破碎的,超声可以破碎菌体释放里面的包涵体,但是不能破碎包涵体;但如果用水煮的话,包涵体会变性,会有一部分可溶于水,所以你跑的上清中有可能有包涵体存在,也有可能没有包涵体; 建议: 还是先将菌体超声破碎,然后离心,取沉淀和上清再跑一次电泳,如果沉淀上清中都有你要的蛋白,说明表达的结果是部分可溶;如果仅上清有就是可溶性表达;如果仅沉淀中有,就是完全包涵体了。不过,一般情况下,应该是第一者的可能性大。

对翻译中异化法与归化法的正确认识

对翻译中异化法与归化法的正确认识 班级:外语学院、075班 学号:074050143 姓名:张学美 摘要:运用异化与归化翻译方法,不仅是为了让读者了解作品的内容,也能让读者通过阅读译作,了解另一种全新的文化,因为进行文化交流才是翻译的根本任务。从文化的角度考虑,采用异化法与归化法,不仅能使译文更加完美,更能使不懂外语的人们通过阅读译文,了解另一种文化,促进各民族人们之间的交流与理解。翻译不仅是语言符号的转换,更是跨文化的交流。有时,从语言的角度所作出的译文可能远不及从文化的角度所作出的译文完美。本文从翻译策略的角度,分别从不同时期来说明人们对异化法与归化法的认识和运用。 关键词:文学翻译;翻译策略;异化;归化;辩证统一 一直以来,无论是在我国还是在西方,直译(literal translation)与意译(liberal translation)是两种在实践中运用最多,也是被讨论研究最多的方法。1995年,美籍意大利学者劳伦斯-韦努蒂(Lawrence Venuti)提出了归化(domestication)与异化(foreignization)之说,将有关直译与意译的争辩转向了对于归化与异化的思考。归化与异化之争是直译与意译之争的延伸,是两对不能等同的概念。直译和意译主要集中于语言层面,而异化和归化则突破语言的范畴,将视野扩展到语言、文化、思维、美学等更多更广阔的领域。 一、归化翻译法 Lawrwnce Venuti对归化的定义是,遵守译入语语言文化和当前的主流价值观,对原文采用保守的同化手段,使其迎合本土的典律,出版潮流和政治潮流。采用归化方法就是尽可能不去打扰读者,而让作者向读者靠拢(the translator leaves the reader in peace, as much as possible, and moves the author towards him)。归化翻译法的目的在于向读者传递原作的基本精神和语义内容,不在于语言形式或个别细节的一一再现。它的优点在于其流利通顺的语言易为读者所接受,译文不会对读者造成理解上的障碍,其缺点则是译作往往仅停留在内容、情节或主要精神意旨方面,而无法进入沉淀在语言内核的文化本质深处。 有时归化翻译法的采用也是出于一种不得已,翻译活动不是在真空中进行的,它受源语文化和译语文化两种不同文化语境的制约,还要考虑到两种文化之间的

蛋白质的纯化方法

蛋白质纯化的方法 蛋白质的分离纯化方法很多,主要有: (一)根据蛋白质溶解度不同的分离方法 1、蛋白质的盐析 中性盐对蛋白质的溶解度有显著影响,一般在低盐浓度下随着盐浓度升高,蛋白质的溶解度增加,此称盐溶;当盐浓度继续升高时,蛋白质的溶解度不同程度下降并先后析出,这种现象称盐析,将大量盐加到蛋白质溶液中,高浓度的盐离子(如硫酸铵的SO4和NH4)有很强的水化力,可夺取蛋白质分子的水化层,使之“失水”,于是蛋白质胶粒凝结并沉淀析出。盐析时若溶液pH在蛋白质等电点则效果更好。由于各种蛋白质分子颗粒大小、亲水程度不同,故盐析所需的盐浓度也不一样,因此调节混合蛋白质溶液中的中性盐浓度可使各种蛋白质分段沉淀。 影响盐析的因素有:(1)温度:除对温度敏感的蛋白质在低温(4度)操作外,一般可在室温中进行。一般温度低蛋白质溶介度降低。但有的蛋白质(如血红蛋白、肌红蛋白、清蛋白)在较高的温度(25度)比0度时溶解度低,更容易盐析。(2)pH值:大多数蛋白质在等电点时在浓盐溶液中的溶介度最低。(3)蛋白质浓度:蛋白质浓度高时,欲分离的蛋白质常常夹杂着其他蛋白质地一起沉淀出来(共沉现象)。因此在盐析前血清要加等量生理盐水稀释,使蛋白质含量在2.5-3.0%。 蛋白质盐析常用的中性盐,主要有硫酸铵、硫酸镁、硫酸钠、氯化钠、磷酸钠等。其中应用最多的硫酸铵,它的优点是温度系数小而溶解度大(25度时饱和溶液为4.1M,即767克/升;0度时饱和溶解度为3.9M,即676克/升),在这一溶解度范围内,许多蛋白质和酶都可以盐析出来;另外硫酸铵分段盐析效果也比其他盐好,不易引起蛋白质变性。硫酸铵溶液的pH常在4.5-5.5之间,当用其他pH值进行盐析时,需用硫酸或氨水调节。 蛋白质在用盐析沉淀分离后,需要将蛋白质中的盐除去,常用的办法是透析,即把蛋白质溶液装入秀析袋内(常用的是玻璃纸),用缓冲液进行透析,并不断的更换缓冲液,因透析所需时间较长,所以最好在低温中进行。此外也可用葡萄糖凝胶G-25或G-50过柱的办法除盐,所用的时间就比较短。

动物微生物实验仪器操作规程汇总(DOC)

高压蒸汽灭菌锅操作规程 一、操作步骤 1、开盖:向左转动手轮数圈,直至转动到顶,使锅盖充分提起,拉起左立柱上的保险销,向右推开横梁移开锅盖。 2、通电:接通电源,此时欠压蜂鸣器响,显示本机锅内无压力(当锅内压力升至约0.03Mpa 时蜂鸣器自动关闭),控制面板上的低水位灯亮,锅内属断水状态。 3、加水:将纯水或生活用水直接注入蒸发锅内约8升,同时观察控制面板上的水位灯,当加水至低水位灯灭,高水位灯亮时停止加水。当加水过多发现内胆有存水,开启下排汽阀放去内胆中的多余水量。 4、放样:将灭菌物品仪器堆放在灭菌筐内,各包之间留有间隙,有利于蒸气的穿透,提高灭菌效果。密封:把横梁推向左立柱内,横梁必须全部推入立柱槽内,手动保险销自动下落锁住横梁,旋紧锅盖。 5、设定温度和时间:按一下确认键,进入温度设定状态,按上下键可以调节温度值,再次按下确认键,进入时间设定状态,按左键或上下键设置需要的时间,再次按动确认键,设定完成,仪器进入工作状态,开始加热升温。 6、灭菌结束后,关闭电源,待压力表指针回落零位后,开启安全阀或排汽排水总阀,放净灭菌室内余气。若灭菌后需迅速干燥,须打开安全阀或排汽排水总阀,让灭菌器内的蒸汽迅速排出,使物品上残留水蒸气快速挥发。灭菌液体时严禁使用干燥方法。 7、启盖:同第一步。 二、注意事项 1、堆放灭菌包时应注意安全阀放汽孔位置必须留出空气,保障其畅通,否则易造成锅体爆裂事故。 2、灭菌液体时,应将液体灌装在耐热玻璃瓶中,以不超过3/4体积为好,瓶口选用棉花纱塞。 3、本器尽量使用纯水,以防产生水垢。 动物微生物生实训室

生物显微镜操作规程 一、操作步骤 1、将所需观察的标本放在工作台上卡夹住。 2、将各倍率物镜顺序装于物镜转换器上,目镜插入目镜筒中。 3、操作时将标本移动到工作台中间,先用10×物镜观察,打开电源开关把亮度调节钮移至适当位置,转动粗调手轮将工作台上升到能见到标本的影形,转动微调手轮即可得到清晰的物象。光亮的选择可转动聚光镜架手轮使聚光镜上升或下降,再调节可变光栏,使改变交栏孔径以便获得适合各类细节标本的照明亮度。(为光源和观察需要备有滤色片供使用,滤色片装于可变光栏下部的托架上,可得到选择的色泽。如用低倍物镜观察液体及用高位物镜时感到光源太强时,可将毛玻片装于可变光栏下部托架上使用,可得到暗淡光线)转动工作台上纵向手轮,使工作台同标本作前后方向移动,转动横向手轮使标本作左右方向移动。将所需观察的物体移至中心观察,然后转至高倍物镜或油浸物镜进行观察(用油镜时需加注香切片物体)仍能看见物体的影象,需再转动微调手轮即可达到清晰的物象。例用完毕只要转动粗调手轮将工作台下降到底,再将亮度调节钮移到最小亮度处最后关上电源开关。 4、调节亮度调节钮可以改变灯泡发光亮度以获得最佳亮度。 5、更换灯泡方法:把钨卤素灯插入灯座,然后将它插入底座下方插口处即可。

(整理)包涵体的分离纯化.

包涵体的纯化和复性总结(二) 关于包涵体的纯化是一个令人头疼的问题,包涵体的复性已经成为生物制药的瓶颈,关于包涵体的处理一般包括这么几步:菌体的破碎、包涵体的洗涤、溶解、复性以及纯化,内容比较庞杂 一、菌体的裂解 1、怎样裂解细菌? 细胞的破碎方法 1.高速组织捣碎:将材料配成稀糊状液,放置于筒内约1/3体积,盖紧筒盖,将调速器先拨至最慢处,开动开关后,逐步加速至所需速度。此法适用于动物内脏组织、植物肉质种子等。 2.玻璃匀浆器匀浆:先将剪碎的组织置于管中,再套入研杆来回研磨,上下移动,即可将细胞研碎,此法细胞破碎程度比高速组织捣碎机为高,适用于量少和动物脏器组织。 3.超声波处理法:用一定功率的超声波处理细胞悬液,使细胞急剧震荡破裂,此法多适用于微生物材料,用大肠杆菌制备各种酶,常选用50-100毫克菌体/毫升浓度,在1KG至10KG 频率下处理10-15分钟,此法的缺点是在处理过程会产生大量的热,应采取相应降温措施,时间以及超声间歇时间、超声时间可以自己调整,超声完全了菌液应该变清亮,如果不放心可以在显微镜下观察。对超声波及热敏感的蛋白和核酸应慎用。 4.反复冻融法:将细胞在-20度以下冰冻,室温融解,反复几次,由于细胞内冰粒形成和剩余细胞液的盐浓度增高引起溶胀,使细胞结构破碎。 5.化学处理法:有些动物细胞,例如肿瘤细胞可采用十二烷基磺酸钠(SDS)、去氧胆酸钠等细胞膜破坏,细菌细胞壁较厚,可采用溶菌酶处理效果更好,我用的浓度一般为1mg/ml。无论用哪一种方法破碎组织细胞,都会使细胞内蛋白质或核酸水解酶释放到溶液中,使大分子生物降解,导致天然物质量的减少,加入二异丙基氟磷酸(DFP)可以抑制或减慢自溶作用;加入碘乙酸可以抑制那些活性中心需要有疏基的蛋白水解酶的活性,加入苯甲磺酰氟化物(PMSF)也能清除蛋白水解酶活力,但不是全部,而且应该在破碎的同时多加几次;另外,还可通过选择pH、温度或离子强度等,使这些条件都要适合于目的物质的提取。 这是标准配方: 裂解液:50mM Tris-HCl(pH8.5~9.0), 2mM EDTA, 100mM NaCl, 0.5% Triton X-1 00, 1mg/ml溶菌酶。(溶菌酶在这个pH范围内比较好发挥作用) 但我个人的经验是:如果你裂解细菌是为了提取蛋白的话,而且蛋白的分子量又小于20kd的话,尽量减少溶菌酶的用量,会引入溶菌酶这种杂蛋白.一般配60ml裂解液用药匙匙柄盛一点就够.判断裂解好坏的标准是,溶液很粘. protocol是10ml-50ml缓冲液(菌体洗涤液,裂解液等)/1g湿菌体. 如果只做一个鉴定,我觉得100-200ml菌就够了. 但凡超声,我都用60ml裂解液,因为我们的超声仪(现代分子生物学实验技术录象里的那种)很适合用100ml小烧杯,装60ml裂解液,这样能让超声头离液面不高不低,不会冒泡泡,也不会洒出来.菌多我就延长超声时间. 沉淀,也就是包涵体沉淀了,如果要上柱纯化,一定要先用4M尿素洗涤一下再用8M尿素溶解.如果不上柱,只是跑跑电泳,可以直接用8M尿素溶解以后,离心取上清,加入适量体积的load ing buffer.loading buffer对于包涵体的溶解能力是较弱的. "取200微升菌液,离心后直接加上样buffer,100度3分钟后上样,然后SDSPAGE. 这个方法到底能不能溶解细菌中的包涵体? " 而楼主的问题,虽然loading buffer对于包涵体的溶解能力是较弱的,但是我觉得你的做法只是在鉴定有无表达,用loading buffer是没有问题的.

常用实验动物

常用实验动物 1、小鼠 喜欢群居,怕热,高温容易中暑 雌雄性小鼠交配后10~12小时,在雌性小鼠阴道口会形成白色的阴道栓 主要解剖学特性 消化系统:食管内壁有角质化鳞状上皮,利于灌胃;有胆囊;胰腺分散,色淡红,似脂肪组织。 生殖系统:雌性小鼠为双子宫型,呈“Y”形,卵巢不予腹腔相通,无宫外孕。 骨髓为红骨髓,无黄骨髓,终身造血。 皮肤无汗腺。 小鼠常用品种、品系 1. 近交系小鼠 C3H:1975年从美国引进,野生色毛; ? C57BL/6:1975年从日本引进,黑色毛; ? BALB/c:Bagg1913年获得小鼠白化株,经近亲繁殖20代以上育成,毛色为白色; ? DBA:分为DBA/1和DBA/2两个品系,1977年由美国实验动物中心引进,毛色均为浅灰色。 2、封闭群小鼠 ①KM小鼠:我国使用量最大的远交种小鼠,白色,抗病、适应力强,繁殖、成活率高。 ②ICR:1973年由日本国立肿瘤研究所引入我国,白色,其显著特点是繁殖

力强。 ③LACA :1973年由英国实验动物中心引入我国,白色。其实是小鼠改名而成。 ④NIH :由美国国立卫生研究院培育,白色,繁殖力强,幼仔成活率高,雄性好斗 3、突变系 1、裸鼠:第11对染色体上的裸基因(nu)导致无毛裸体、无胸腺 2、SCID小鼠:第16对染色体上的Scid隐性基因突变基因导致T、B淋巴细胞联合免疫缺陷.外观与普通小鼠差别不大,被毛白色,体重发育正常 3、快速老化模型小鼠:4~6月龄以前与普通小鼠的生长一样,4~6月龄以后迅速出现老化症状。如心、肺、脑、皮肤等器官老化,出现骨质疏松和老化淀粉样变。侏儒症:比正常小鼠体型小,缺少生长素和促甲状腺素,用于内分泌研究。小鼠在医学、生物学的应用 1、重组近交系小鼠将双亲品系的基因自由组合和重组产生一系列的子系,是遗传分析的重要依据,用作基因定位及其连锁关系的研究 2、提供自然的动物模型 2、大鼠染色体数2n=42 喜独居,喜啃咬,性情较凶猛、抗病力强,对新环境适应力强,但对环境刺激、炎症反应敏感。强烈噪音可引起食仔或抽搐;湿度低于40%易发生环尾巴症。行为表现多样,情绪反应敏感,易接受通过正负强化进行的多种感觉指令的训练雌性2.5月龄达到性成熟,具有产后发情、产后妊娠的特点。寿命2.5~3年。解剖学特征

标准操作规程实验动物隔离检疫标准操作规程【模板】

标 准 操 作 规 程 (Standard Operating Procedure) 实验动物隔离检疫 标准操作规程 Standard Operating Procedure for ethical review system of Laboratory Animal 制定者 Author 兽医办公室/兽医师 Veterinary office 审查者 Reviewer 肖春兰 动物质量办公室/技术员 Technican of Animal Quality Lab 审查者 Reviewer 王婧 办公室主任 Office Director 机构负责人批准Facility Manager Approver 周正宇 中心最高领导者 Top Manager of the Center

修订记录(Revision History)

发放记录(Revision History)

1、目的(Purpose) 规范苏州大学实验动物中心实验动物隔离检疫标准操作规程。 2、适用范围 (Scope) 此文件适合苏州大学实验动物中心所有动物隔离检疫。 3、职责(Responsibility) 3.1 文件编写、批准人员对该文件编写和修改的有效性负责。 3.2 文件编写人员负责根据此文件,对担任实验动物隔离检疫的工作人进行 培训和指导。 3.3 担任实验动物隔离检疫的工作人,负责执行本标准操作规程,并负责反馈 相关信息。 4、操作规程(Procedures) 凡自主采购且来源为不是免检单位(见附表1)的SPF级实验动物,均需在进入我中心SPF动物房前,接受隔离检疫。 4.1 提交自购申请 凡自主采购实验动物的个人及单位,均需向我中心提交自购实验动物申请。 4.1.1 登陆动物管理系统 科研计划人以科研计划用户身份登陆苏州大学实验动物管理系统。 4.1.2 提交动物订购申请 科研计划人点击动物订购项,添加动物订购申报。 4.1.3 注意事项

包涵体纯化全过程(3)

一、包涵体的纯化和复性总结(二) 二、[ 2008-2-25 14:25:00 | By: 飞鸿 ] 三、关于包涵体的纯化是一个令人头疼的问题,包涵体的复性已经成为生物制药的瓶颈,关于包涵体的处理一般包括这么几步:菌体的破碎、包涵体的洗涤、溶解、复性以及纯化,内容比较庞杂 一、菌体的裂解 1、怎样裂解细菌? 细胞的破碎方法 1.高速组织捣碎:将材料配成稀糊状液,放置于筒内约1/3体积,盖紧筒盖,将调速器先拨至最慢处,开动开关后,逐步加速至所需速度。此法适用于动物内脏组织、植物肉质种子等。 2.玻璃匀浆器匀浆:先将剪碎的组织置于管中,再套入研杆来回研磨,上下移动,即可将细胞研碎,此法细胞破碎程度比高速组织捣碎机为高,适用于量少和动物脏器组织。 3.超声波处理法:用一定功率的超声波处理细胞悬液,使细胞急剧震荡破裂,此法多适用于微生物材料,用大肠杆菌制备各种酶,常选用50-100毫克菌体/毫升浓度,在1KG至10KG频率下处理10-15分钟,此法的缺点是在处理过程会产生大量的热,应采取相应降温措施,时间以及超声间歇时间、超声时间可以自己调整,超声完全了菌液应该变清亮,如果不放心可以在显微镜下观察。对超声波及热敏感的蛋白和核酸应慎用。 4.反复冻融法:将细胞在-20度以下冰冻,室温融解,反复几次,由于细胞内冰粒形成和剩余细胞液的盐浓度增高引起溶胀,使细胞结构破碎。 5.化学处理法:有些动物细胞,例如肿瘤细胞可采用十二烷基磺酸钠(SDS)、去氧胆酸钠等

细胞膜破坏,细菌细胞壁较厚,可采用溶菌酶处理效果更好,我用的浓度一般为1mg/ml。无论用哪一种方法破碎组织细胞,都会使细胞内蛋白质或核酸水解酶释放到溶液中,使大分子生物降解,导致天然物质量的减少,加入二异丙基氟磷酸(DFP)可以抑制或减慢自溶作用;加入碘乙酸可以抑制那些活性中心需要有疏基的蛋白水解酶的活性,加入苯甲磺酰氟化物(PMSF)也能清除蛋白水解酶活力,但不是全部,而且应该在破碎的同时多加几次;另外,还可通过选择pH、温度或离子强度等,使这些条件都要适合于目的物质的提取。 这是标准配方: 裂解液:50mM Tris-HCl(pH8.5~9.0), 2mM EDTA, 100mM NaCl, 0.5% Triton X-100, 1mg/ml 溶菌酶。(溶菌酶在这个pH范围内比较好发挥作用) 但我个人的经验是:如果你裂解细菌是为了提取蛋白的话,而且蛋白的分子量又小于20kd的话,尽量减少溶菌酶的用量,会引入溶菌酶这种杂蛋白.一般配60ml裂解液用药匙匙柄盛一点就够.判断裂解好坏的标准是,溶液很粘. protocol是10ml-50ml缓冲液(菌体洗涤液,裂解液等)/1g湿菌体. 如果只做一个鉴定,我觉得100-200ml菌就够了. 但凡超声,我都用60ml裂解液,因为我们的超声仪(现代分子生物学实验技术录象里的那种)很适合用100ml小烧杯,装60ml裂解液,这样能让超声头离液面不高不低,不会冒泡泡,也不会洒出来.菌多我就延长超声时间. 沉淀,也就是包涵体沉淀了,如果要上柱纯化,一定要先用4M尿素洗涤一下再用8M尿素溶解.如果不上柱,只是跑跑电泳,可以直接用8M尿素溶解以后,离心取上清,加入适量体积的loading buffer.loading buffer对于包涵体的溶解能力是较弱的. "取200微升菌液,离心后直接加上样buffer,100度3分钟后上样,然后SDSPAGE. 这个方法

蛋白纯化(his标签)说明书

Instruction Manual ProBond TM Purification System For purification of polyhistidine-containing recombinant proteins Catalog nos. K850-01, K851-01, K852-01, K853-01, K854-01, R801-01, R801-15 Version K 2 September2004 25-0006

ii

Table of Contents Kit Contents and Storage (iv) Accessory Products (vi) Introduction (1) Overview (1) Methods (2) Preparing Cell Lysates (2) Purification Procedure—Native Conditions (7) Purification Procedure—Denaturing Conditions (11) Purification Procedure—Hybrid Conditions (13) Troubleshooting (15) Appendix (17) Additional Protocols (17) Recipes (18) Frequently Asked Questions (21) References (22) Technical Service (23) iii

Kit Contents and Storage Types of Products This manual is supplied with the following products: Product Catalog No. ProBond? Purification System K850-01 ProBond? Purification System with Antibody with Anti-Xpress? Antibody K851-01 with Anti-myc-HRP Antibody K852-01 with Anti-His(C-term)-HRP Antibody K853-01 with Anti-V5-HRP Antibody K854-01 ProBond? Nickel-Chelating Resin (50 ml) R801-01 ProBond? Nickel Chelating Resin (150 ml) R801-15 ProBond?Purification System Components The ProBond? Purification System includes enough resin, reagents, and columns for six purifications. The components are listed below. See next page for resin specifications. Component Composition Quantity ProBond? Resin 50% slurry in 20% ethanol 12 ml 5X Native Purification Buffer 250 mM NaH2 PO4, pH 8.0 2.5 M NaCl 1 × 125 ml bottle Guanidinium Lysis Buffer 6 M Guanidine HCl 20 mM sodium phosphate, pH 7.8 500 mM NaCl 1 × 60 ml bottle Denaturing Binding Buffer 8 M Urea 20 mM sodium phosphate, pH 7.8 500 mM NaCl 2 × 125 ml bottles Denaturing Wash Buffer 8 M Urea 20 mM sodium phosphate, pH 6.0 500 mM NaCl 2 × 125 ml bottles Denaturing Elution Buffer 8 M Urea 20 mM NaH2PO4, pH 4.0 500 mM NaCl 1 × 60 ml bottle 3 M Imidazole, 20 mM sodium phosphate, pH 6.0 500 mM NaCl 1 × 8 ml bottle Purification Columns 10 ml columns 6 Continued on next page iv

动物实验标准操作规程

动物实验标准操作规程 一、人流线路工作人员进入第一更衣室→脱去个人外衣(饰品)放入衣柜→脱去蓝色拖鞋后(→淋浴室)进入第二更衣室→穿上红色拖鞋→手消毒(0.1%新洁尔灭)→按操作规范穿上无菌连体衣,戴上一次性无菌口罩、手套→进入缓冲间→风淋→进入清洁走廊→进入洁净区域工作→进入饲养室或检疫室→所有工作结束后→进入污物走廊、脱掉红色拖鞋→进入缓冲间→穿上蓝色拖鞋,进入洗刷、消毒区域,取掉手套、口罩、连体衣,分别放入指定的回收桶中→进入第一更衣室,穿上个人衣(饰)→出屏障系统。 二、物流线路物品→高压蒸汽灭菌器或传递窗或渡槽→消毒传递间→清洁准备室→清洁走廊→饲养室或动物实验室→污物经包装处理→(后室缓冲间)→次清洁走廊→气闸(缓冲间)→清洗消毒室→外部区域。 三、动物流线路外来SPF级实验动物→传递窗→消毒传递间→清洁准备室→清洁走廊→饲养室或动物实验室→生产繁殖或实验处理后→(后室缓冲间)→次清洁走廊→气闸(缓冲间)→清洗消毒室→外部区域。 四、空气(压力)流程新风口→空气初效过滤器→空气中效过滤器→空气高效过滤器→屏障系统内各区域并产生压力梯度: ①清洁走廊→饲养室或动物实验室→(后室缓冲间)→次清洁走廊→气闸(缓冲间)→室外。②(进入屏障系统内)气闸(缓冲间)→第2更衣室→淋浴室→第1更衣室→室外。③(离开屏障系统内)气闸(缓冲间)→清洗准备间

(消毒室)→外部区域。④消毒传递间→传递窗→清洗消毒室→外部区域。 五、注意事项为了防止空气倒流,必须调节并保持4.4中各区域间的压力梯度,同时,必须时刻保证2更衣室、消毒传递间、气闸(缓冲间)至少20Pa的正压。 六、质量记录动物/物品传递管理记录表、动物饲养环境参数与异常情况记录表。

蛋白纯化的一般原则及方法选择

随着分子生物学的发展,越来越多的科研人员熟练掌握了分子生物学的各种试验技术,并研制成套试剂盒,使基因克隆表达变得越来越容易lIl。但分子生物学的上游工作往往并非是最终目的,分子克隆与表达的关键是要拿到纯的表达产物,以研究其生物学作用,或者大量生产出可用于疾病治疗的生物制品。相对与上游工作来说,分子克隆的下游工作显得更难,蛋白纯化工作非常复杂,除了要保证纯度外,蛋白产品还必须保持其生物学活性。纯化工艺必须能够每次都能产生相同数量和质量的蛋白,重复性良好。这就要求应用适应性非常强的方法而不是用能得到纯蛋白的最好方法去纯化蛋白。在实验室条件下的好方法却可能在大规模生产应用中失败,因为后者要求规模化,且在每日的应用中要有很好的重复性。本文综述了蛋白质纯化的基本原则和各种蛋白纯化技术的原理、优点及局限性,以期对蛋白纯化的方法选择及整体方案的制定提供一定的指导。 1 蛋白纯化的一般原则 蛋白纯化要利用不同蛋白间内在的相似性与差异,利用各种蛋白间的相似性来除去非蛋白物质的污染,而利用各蛋白质的差异将目的蛋白从其他蛋白中纯化出来。每种蛋白间的大小、形状、电荷、疏水性、溶解度和生物学活性都会有差异,利用这些差异可将蛋白从混合物如大肠杆菌裂解物中提取出来得到重组蛋白。蛋白的纯化大致分为粗分离阶段和精细纯化阶段二个阶段。粗分离阶段主要将目的蛋白和其他细胞成分如DNA、RNA等分开,由于此时样本体积大、成分杂,要求所用的树脂高容量、高流速,颗粒大、粒径分布宽.并可 以迅速将蛋白与污染物分开,防止目的蛋白被降解。精细纯化阶段则需要更高的分辨率,此阶段是要把目的蛋白与那些大小及理化性质接近的蛋白区分开来,要用更小的树脂颗粒以提高分辨常用的离子交换柱和疏水柱,应用时要综合考虑树脂的选择性和柱效两个因素。选择性指树脂与目的蛋白结合的特异性,柱效则是指蛋白的各成分逐个从树脂上集中洗脱的能力,洗脱峰越窄,柱效越好。仅有好的选择性,洗脱峰太宽,蛋白照样不能有效分离。 2.各种蛋白纯化方法及优缺点 2.1蛋白沉淀蛋白能溶于水是因为其表面有亲水性氨基酸。在蛋白质的等电点处若溶液的离子强度特别高或特别低,蛋白则倾向于从溶液中析出。硫酸铵是沉淀蛋白质最常用的盐,因为它在冷的缓冲液中溶解性好,冷的缓冲液有利于保护蛋白的活性。硫酸铵分馏常用做纯化的第一步,它可以初步粗提蛋白质,去除非蛋白成分。蛋白质在硫酸铵沉淀中较稳定,可以短期在这种状态下保存中间产物,当前蛋白质纯化多采用这种办法进行粗分离翻。在规模化生产上硫酸铵沉淀方法仍存在一些问题,硫酸铵对不锈钢器具的腐蚀性很强。其他的盐如硫酸钠不存在这种问题,但其纯化效果不如硫酸铵。除了盐析外蛋白还可以用多聚物如PEG 和防冻剂沉淀出来,PEG是一种惰性物质,同硫酸铵一样对蛋白有稳定效果, 在缓慢搅拌下逐渐提高冷的蛋白溶液中的PEG浓度,蛋白沉淀可通过离心或过滤获得,蛋白可在这种状态下长期保存而不损坏。蛋白沉淀对蛋白纯化来说并不是多么好的方法,因为它只能达到几倍的纯化效果,而我们在达到目的前需要上千倍的纯化。其好处是可以把蛋白从混杂有蛋白酶和其他有害杂质的培养基及细胞裂解物中解脱出来。

翻译中的归化与异化

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抗肿瘤药物体内筛选试验标准操作规程(SOP)

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按照急性毒性测试的常规方法,选用昆明种小鼠,通过腹腔注射方式给药,测定体外抗肿瘤活性突出的化合物的半数致死量(LD50),参考给药小鼠体重变化情况,评价化合物的急性毒性,并确定小鼠体内抗肿瘤活性测试的给药剂量。 (b)小鼠体内抗肿瘤活性测试 根据动物体内抗肿瘤活性测试的标准方法,选用昆明种小鼠,皮下接种肉瘤S180或肺癌H22瘤株,选择体外活性突出且急性毒性较低的化合物,设定合适的剂量通过腹腔注射方式给药,以临床常用抗肿瘤药物环磷酰胺作为阳性对照药物,测定肿瘤生长抑制作为体内活性评价指标。 (c)专利保护范围内的化合物的继续合成 申请保护范围较大的专利,合成部分可能具有良好活性的新的化合物,拓展研究范围,发现活性更强的化合物,并申请新的发明专利。并可针对具体化合物申请从属专利,延长高活性化合物的保护期限。 (d)体外抗肿瘤活性的广泛筛选 采用MTT法或台盼蓝染色法,测定化合物对多种人肿瘤细胞株的增殖抑制活性,确定化合物在不同瘤株间抗肿瘤活性的选择性,为裸鼠模型实验提供依据。 (e)抗肿瘤作用机理的深入研究 根据抗肿瘤(f)人癌裸鼠移植瘤模型实验活性化合物作用机理特征,选用微管蛋白聚合等实验从分子水平确认化合物的作用机理;利用人脐静脉血管内皮细胞探讨化合物对内皮细胞骨架的影响及诱导凋亡的途经,从细胞水平上阐明化合物的作用机理。 根据抗肿瘤新药审批办法的要求,采用裸小鼠皮下接种模型和/或原位移植瘤模型,以相对肿瘤增值率和生存时间为指标,确定化合物的抗肿瘤活性。 (g)动物体内药物代谢动力学实验

包涵体蛋白的分离纯化

包涵体蛋白的分离纯化 赵玲0743085096 包涵体是外源基因在原核细胞中表达时,尤其在大肠杆菌中高效表达时,形成的由膜包裹的高密度、不溶性蛋白质颗粒,在显微镜下观察时为高折射区,与胞质中其他成分有明显区别。包涵体形成是比较复杂的,与胞质内蛋白质生成速率有关,新生成的多肽浓度较高,无充足的时间进行折叠,从而形成非结晶、无定形的蛋白质的聚集体;此外,包涵体的形成还被认为与宿主菌的培养条件,如培养基成分、温度、pH 值、离子强度等因素有关。细胞中的生物学活性蛋白质常以可融性或分子复合物的形式存在,功能性的蛋白质总是折叠成特定的三维结构型。包涵体内的蛋白是非折叠状态的聚集体,不具有生物学活性,因此要获得具有生物学活性的蛋白质必须将包涵体溶解,释放出其中的蛋白质,并进行蛋白质的复性。包涵体的主要成分就是表达产物,其可占据集体蛋白的40%~95%,此外,还含有宿主菌的外膜蛋白、RNA聚合酶、RNA、DNA、脂类及糖类物质,所以分离包涵体后,还要采用适当的方法(如色谱法)进行重组蛋白质的纯化。 1. 包涵体的形成 重组蛋白不论在原核细胞还是真核细胞中表达时,都可形成包涵体。通常所说的包涵体是指重组蛋白在大肠杆菌中高效表达时形成的无活性蛋白聚集体,一般含有50%以上重组蛋白,其余为核糖体组分、RNA聚合酶,外膜蛋白等杂蛋白,以及质粒DNA、RNA片断、脂质、肽聚糖、脂多糖等成分]。由于包涵体在相差显微镜下为黑色斑点, 所以也称为折射体。包涵体形成的原因主要有以下几点: ⑴蛋白合成速度太快,以致于没有足够的时间进行折叠。蛋白折叠的动力学模型表明:蛋白质天然构象形成的速率取决于肽链的合成速率、折叠速率和聚集速率几个因素。中间体正确折叠是分子内的一级反应,而中间体的聚集是发生在分子间的二级或高级反应,因此,折叠中间体的浓度对聚集反应影响非常大];⑵重组蛋白是大肠杆菌的异源蛋白,由于缺少真核生物的翻译后修饰系统(如糖基化等) ,致使中间体大量积累,容易形成包涵体;⑶培养条件不佳和重组蛋白所处的环境也可导致包涵体形成,如发酵温度高,胞内pH 接近蛋白的等电点等;⑷二硫键在蛋白折叠中有重要作用,而大肠杆菌胞内的还原环境不利于二硫键的形成;⑸包 涵体不溶可能由于分子间无活性的β2片层含量高于天然结构或盐沉淀蛋白。包涵体蛋白虽然不具有天然

翻译的归化与异化

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