rev: October 28, 1999
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General Western Blot Protocol
Below find a generalized western blot protocol that can be used with many of our antibodies. There is no "one" perfect protocol and of course each antibody must be optimized. See each antribody spec sheet for references and additional suggested dilutions. With the introduction of enhanced chemiluminescence detection, many antibodies can first be tried at 0.5ug/ml with overnight incubaton and then titered from that point. The general working range for most antibodies seems to fall from 2ug/ml to 0.1ug/ml.
-if you have a favorite protocol when using any of our antibodies or a generalized protocol, please send us a copy and we will add it to this list to help other reserachers.
Sample Western Blotting Protocol: METHOD A
-prepare samples in cold RIPA (3.0ml/gram tissue, or 1ml per 20 million cells) and add fresh inhibitors 10-30ul of 10mg/ml PMSF stock per gram tissue or 20 million cells). Clarify by centrifugation 10,000 X G for 10 minutes at 4 DEG C. The supernatant fluid is the total cell lysate. Additional
General Sample Prep: Must be optimized for each method:
a) Tissue samples:
-weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue can be sliced very thinly and thawed in lysis buffer containing inhibitors. Use 3ml of ice cold RIPA buffer per gram of tissue. -Further disrupt and homogenize tissue with a dounce homogenizer or a polytron device, maintaining temperature at 4 DEG C throughout all procedures. Add 30ul of 10mg/ml PMSF stock per gram of tissue and incubate on ice for 30 minutes.
-Transfer to microcentrifuge tubes, centrifuge at 10,000 X g for 10 minutes at 4 DEG C. Remove supernatant and centrifuge again. The supernatant fluid is the total cell lysate. Sometimes a longer centrifugation is necessary to obtain a clarified lysate.
1) 1) Mix sample** (50-100ug whole cell lysate, 10-20ug nuclear extract or 10-20ng purified protein per lane ) with an equal volume of 2X electrophoresis sample buffer and boil for 2-3 minutes. Unused samples may be able to be stored at -20 DEG C for up to several months.
** Amounts of sample may need to be increased for low prevalent material
or decreased when more sensitive detection systems are available. Must be
determined experimentally for all conditions,
2) Load up to 10ul of lysate per 1.0mm of well width for gels of 0.75mm thickness. Load molecular weight markers as per manufactures instructions.
3) Carry out electrophoresis according to standard protocols
4) Transfer from the gel to nitrocellulose membrane using an electroblotter apparatus according to the manufacturer's protocols or by wet transfer (1 ampere for 1 hour using buffer 25mM Tris, 190mM glycine, 20% MeoH). NOTE: in some cases, high molecular weight proteins, ie >120kDa may require 90 minutes transfer time in wet systems and require the addition of 0.05% SDS to the transfer buffer. The transferred proteins may be able to be visualized by staining the membarnes for 15-30 minutes with india ink (Higgin black india ink-Eberhard Faber), diluted 1:1000 in wash buffer (10mM Tris pH 7.5, 100mM NaCl, 0.1% Tween). Rinse excess stain before blocking.
5) Block non specific binding by soaking membrane in suitable protein buffer for at least 30 minutes at room temperature. Alternatively, the membrane may be blocked at 4 DEG C overnight in a covered container using Blocking protein Buffer without Tween. Note:With Biotin detection systems, the use of a good grade of BSA (2%) or (1-2%) biotin free casein is suggested (some grades of skim milk contain endogeneous biotin). Skim milk can also be problematic with phosphotyrosine/serine or other phosphspecific antibodies.
6) Incubate with primary antibody at 0.1ug/ml to 1.0ug/ml in Blocking Buffer for at least 1 hour at room temp (or overnight at 4 DEG C for maximum detection). Wash three times for 5 minutes each with TBS, 0.05% Tween 20. Note: to spare antibody, an initial test at 0.5ug/ml is a good starting point. Note: if high backgrounds are experienced, try increasing washing time for 6 X, changing bufer every 5 minutes.
7). 7). Incubate for 30-60 minutes with HRP conjugated secondary antibody or alkaline phosphatase conjugated secondary antibody diluted 1:500 to 1:5000 (see manufacturers recommended concentration, initially use at most concentrated dilution recommended by manufacturer, if high backgrounds occur, secondary antibody should be diluted further). Note: If using a polyclonal on a similar species, the use of affinity purified and absorbed antibodies may reduce non specific bands (ie, bands that appear when no primary antibody is used, only the detecting antibody)
-see below for example MW of immunoglobulin bands
8) Wash three time for 5 minutes each with TBS, 0.05% Tween-20 and one additional time for 5 minutes with TBS (no Tween).
9) Incubate with suitable chemiluminescence reagents according to manufacturers protocol.
Solutions: a) TBS=10mM Tris-HCL, pH 8.0, 150mM Nacl
Buffer (2X): 1.0ml glycerol; 0.5ml B-mercaptoethanol; 3.0ml of 10% SDS; 1.25ml
1.0M Tris-HCL pH 6.7; 1-2mg bromophenol blue. Store frozen in small aliquots.
Alternatively make buffer without B-mercaptoethanol and store at room temp.
Add B-mercaptoethanol just before using.
c) Blocking Buffer: 1X TBS, 5% skim milk, 0.05% Tween-20. If using a biotinylated detection system or for special applications (as use of phosphotyrosine antibodies), do not use skim milk, use 2% Biotin free casein or 2% BSA (RIA grade or an extensively dialyzed grade).
d) PBS (1x): 9.1mM dibasic sodium phosphate, 1.7mM monobasic sodium phosphate, 150mM NaCl. Adjust pH to 7.4 with NaOH.
e) RIPA= 1 X PBS, 1% NP40 *, 0.5% sodium deoxycholate, 0.1% SDS (this may be made in large volumes. Add inhibitors at time of use from the following stock solutions).
a) 10mg/ml PMSF in isopropanol (use at 10ul/ml)
b) Aprotinin (Sigma cat#A6279, use at 30ul/ml)
c) 100nM sodium orthovanadate in frozen aliquots (use at 10ul/ml)
* Note:NP-40 is available through United States Biochemical/Amersham. a substitute may also be used=Igepal CA-630 (which is chemicallyindistinguishable from NP40 for most applications). This is available from Sigma Chemical.
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|Non-reduced MW||Reduced MW*|
*numbers ot the left of the comma represent the molecular weight of the heavy chain of immunoglobulins, and numbers to the right are weights for the light chains of immunoglobulins
b= may appear as a doublet on SDS-PAGE
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