Question (43128) | Anti-RED1 antibody (ab89436)

Dear technical support team:

This customer has purchase ab89436 (Anti-RED1 antibody) and has conducted the wb with human sample. The results show the expression was doesn’t decrease of knowdown sample; therefore, she wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?

I also attached her image in this letter and her experiment step as follow:

1. Order details:

Batch number: gr37579-2

Abcamproduct code: ab89436

Antibody storage conditions (temperature/reconstitution etc) 20℃


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

The expression was doesn't decrease of knowdown sample

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

· Species: human

· What’s cell line or tissue: HeLa cell

· Cell extract or Nuclear extract: Cell extract

· Purified protein or Recombinant protein:


3. The lysate

How much protein was loaded:50 ug

What lysis buffer was used: WCE


What protease inhibitors were used: PI (Roche)

What loading buffer was used:

Phosphatase inhibitors :NaF, Na4VO3, souium pyrophosphate, beta-glycerolphosphate

Did you heat the samples: temperature and time:100℃, 10min

4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel:Reducing gel

Reducing agent: DTT

Gel percentage :8%

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


5. Blocking conditions

Buffer: 1XTBST

Blocking agent: milk, BSA,serum, what percentage: 5% non-fat milk

Incubation time: 1 hr

Incubation temperature: RT


6. Primary Antibody

Species: mouse

Reacts against: human

At what dilution(s) have you tested this antibody: 1/1000
What dilution buffer was used: 5% non-fat milk in 1X TBST
Incubation time: O/N
Incubation temperature: 4℃
What washing steps were done: 5 times, 1X TBST

7. Secondary Antibody

Species: goat-anti-mouse IgG

Reacts against:

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

Incubation time: 1 hr

Wash steps: 5 times, 1X TBST

Fluorochrome or enzyme conjugate: HRP

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. Other antibody and siRNA-treated cells

10. Optimization attempts

How many times have you tried the Western?2 times

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

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

What steps have you altered? no

Could you please help this customer to solve the problem?

Thanks for your kindly help

Best regards