Anti-Rac1 antibody [0.T.127] (ab33186)

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ANSWER:

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Dear technical support team:

This customer has purchased ab33186 (Anti-Rac1 antibody [0.T.127]) and has conducted the WB several times in human HEK293T cell line. The results show multiple bands; therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her wb result? I attached the wb image in this letter and her experiment step as follow:

1. Order details:

Batch number: GR44981-1

Abcam product code: ab33186

Antibody storage conditions (temperature/reconstitution etc) Temperature: -20℃

Reconstitution: non

2. Please describe the problem (high background, wrong band size, more bands, no band etc).

High background and more bands

3. On what material are you testing the antibody in WB?

Species: human

What’s cell line or tissue: HEK293T cell line

Cell extract or Nuclear extract: cell extract and cytosol extract

Purified protein or Recombinant protein: non

3. The lysate

How much protein was loaded: 20ug

What lysis buffer was used:2%SDS

What protease inhibitors were used: non

What loading buffer was used: non

Phosphatase inhibitors: non

Did you heat the samples: temperature and time: 95℃ 15mins

4. 

Electrophoresis/Gel conditions/ Transfer conditions

Reducing or non reducing gel: Reducing

Reducing agent: SDS

Gel percentage :12%

Transfer conditions: (Type of membrane, Protein transfer verified):

nitrocellulose, Yes

5. Blocking conditions

Buffer: TBST

Blocking agent: milk, BSA, serum, what percentage:5%milk

Incubation time:1hrs

Incubation temperature: room temperature

6. Primary Antibody

Species: mouse

Reacts against:

At what dilution(s) have you tested this antibody: 1:1000-3000

What dilution buffer was used: 5% milk in TBST

Incubation time: O/N

Incubation temperature: 4℃

What washing steps were done: 4 setps

7. Secondary Antibody

Species: rabbit

Reacts against: mouse

At what dilution(s) have you tested this antibody: 1:5000

Incubation time: 1hrs

Wash steps: 5 steps

Fluorochrome or enzyme conjugate: HRP conjugate

Do you know whether the problems you are experiencing come from the secondary? no

8. Detection method

ECl, ECl+, other detection method:

ECL

9. Did you apply positive and negative controls along with the samples? Please specify. No

10. Optimization attempts

How many times have you tried the Western? 3 times

Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):

non

Do you obtain the same results every time e.g. are background bands always in the same place? yes

What steps have you altered? Concentration of primary antibody

Thanks for your kindly assistance.

Best regards  

ANSWER:

Thank you for taking time to complete the questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimise the results from ab33186, and I have a question in regards to the expression.

-I would recommend using a lysis buffer such as RIPA or Tris-HCl with protease inhibitors added in lysing your cells. More information on lysis buffer preparation can be found here: http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf

-blocking buffers which have previously been used successfully with this antibody are: 5% milk or 3% BSA

-I would recommend trying a lower dilution of the antibody: 1/500

-Although the incubation has been performed overnight at 4℃, I would also recommend trying incubation for 1 hour at room temperature.

-Additionally, carrying out a control leaving out the primary antibody will enable you to see if this non-specificity is as a result of the secondary antibody.

Should the suggestions not improve the results, please do let me know.

I was also wondering if the Rac1 expression was being induced from an expression vector or is it the endogenous expression levels you are trying to detect?

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Dear technical support team,

Our customer raises an enquiry about ab33186 (Anti-Rac1 antibody [0.T.127]), according to the online datasheet this antibody’s positive control can be used rat brain microsomal protein, this customer wants to know is there any suggestion can help she to choice the other positive control? Could you please help this customer to solve the problem? Thanks for your kindly assistance.

Best regards

ANSWER:

Thank you for contacting us.

The other positive control lysates you can use are Hela, MCF7, A431 or Raji cell lysates, or Human tubular colorectal, prostate and squamous cells carcinoma tissue lysates.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Can you recommend a positive control for this antibody?

ANSWER:

Thank you for your enquiry. For Western blotting with this antibody, we recommend obtaining a membrane fraction from rat brains. The following link is for a paper with a protocol for isolating membrane fractions. This procedure will enrich for the Rac1 protein. Consider replacing or adding to the PMSF with a protease inhibitor cocktail:

http://www.jbc.org/cgi/reprint/267/1/467

Another resource is this article which contains an in-depth discussion of microsome fraction preparation:

http://www.bioscience.org/1998/v3/d/heineman/3.htm

I hope this helps. Please do not hesitate to contact us if you have additional questions.